PRODUCTION OF POLYHYDROXYBUTYRATE USING
BACTERIAL STRAINS
Thesis submitted in partial fulfillment of the
requirements for the degree of
Bachelor of technology
In
Biotechnology and Medical Engineering
By
LAXMI BADAIK
111BM0541
UNDER THE SUPERVISION OF
Dr. Nivedita Patra
Department of Biotechnology and Medical Engineering
Department of Biotechnology and Medical Engineering
National Institute of Technology, Rourkela
Rourkela-769008, Odisha, India
Certificate
This to certify that the work in the thesis entitled “PRODUCTION OF
POLYHYDROXYBUTYRATE USING BACTERIAL STRAINS OF Lactobacillus acidophilus” by
Laxmi Badaik (111bm0541) is a record of an original work carried out with my supervision and
guidance in partial fulfillment of the requirements for the degree of Bachelor in Technology in
Biotechnology and Medical Engineering. Neither this thesis nor any part of it has been submitted for any
degree elsewhere.
Dr.Nibedita Patra
Assistant Professor
Department of Biotechnology and Medical Engineering
National Institute of Technology, Rourkela, Odisha-769008
Acknowledgement
Our first thanks are to the almighty, without whose blessings we would not have been this
acknowledgment. I offer my sincerest gratitude to our guide Dr, Nibedita Patra for her stimulating
support. She has always given me opportunity to learn more and more, supported me throughout my
thesis with constant encouragement, being patient. At every point of time she helped me to do better and
guided me with each important and even small thing. I am highly indebted to her for taking out her
precious time for me and guiding me through a proper channel. It would not have been possible this way
without her.
I would also like to thanks to the dr.Indranil banarjee Sir and Dr kunal pal Sir for supporting me in
Laboratory. Above all, I am blessed with such caring parents who have given me full freedom to do
whatever I wish and have always supported me in my decision. I extend my deepest gratitude to my
parents and family members for their invaluable love, affection, encouragement and support.
I would like to thank my friend Ipsita Panda and PhD student(Mr. jay Prakash Yadav)working on that
Lab who have been in one consistent source of motivation and affection and who have in one or another
way have helped me in successful completion of this project.
Laxmi Badaik
111bm0541
CONTENTS
1. Acknowledgment…….
2. Abstract…………
3. Introduction……….
3.1. Lactobacillus acidophilus
4. Methods to find out the yield of PHB
4.1. Gas chromatography
4.2. High performance liquid chromatography
5 .Methods
5.1. Isolation of microbial structure
5.2. Batch cultivation of E.coli cells in a stirred tank bioreactor
5.3. Measurement of cell optical density
5.4. Measurement of cell concentration
5.5. Measurement of glucose concentration
5.6. Estimation of PHB
6. Result
ABSTRACT:
Polyhydroxybutyrate is a polyhydroxyalkanoate (PHA), a polymer having a place with the polyesters
classes. That areinterest as biodegradable and bio-derived plastics. The poly-3-hydroxybutyrate (P3HB)
sort of PHB ispresumably the most widely recognized sort of polyhydroxyalkanoate, yet different
polymers of this class are created by a mixture of life forms: these incorporate poly-4-hydroxybutyrate
(P4HB), polyhydroxyvalerate (PHV), polyhydroxyhexanoate (PHH), polyhydroxyoctanoate (PHO) and
their copolymers.Synbiotic sachets were obtained from general medical store and Soil was gathered
from garden and screened for PHB delivering microscopic organisms. A few unidentified bacterial
colonies were confined utilizing serial dilation method. Every bacterial colony was kept up in slants and
liquid cultures. A lab strain of Lactobacillus was refined and kinetic studies were performed.
Lactobacillus acidophilus produced PHB.
Keyword:
Polyhydroxybutyrate, hydroxybutyrate, Lactobacillus, kinetic studies.
INTRODUCTION
Polyhydroxybutyrate is a polyhydroxualkanoate, which is of a great deal of enthusiasm as it has the
attributes of being a bioplastic. It is biodegradable. Remembering the ecological issues of the era,
polyhydroxybutyrate creation can be of extraordinary use in conveying these issues to a hindered
rate. Polyhydroxybutyrate, not at all like different classes of bioplastics is water insoluble which
others are touchy to dampness. It is impervious to bright beams too. It has the qualities of good
oxygen porousness. It can disintegrate in chlorinated hydrocarbons. It has a softening purpose of
175 degree Celsius. Above all it is non-harmful and biocompatible which makes it suitable to
biomedical applications. Its rigidity is 40 MPa which is near to that of polypropylene. It can be
biodegraded an aerobically.
PHB is naturaly produced by a few microorganisms for the most part in light of anxiety conditions
like supplement constraint. The polymer is principally a result of carbon digestion (from glucose or
starch) and is utilized by microorganisms as a type of vitality stockpiling atom to be metabolized
when other basic vitality sources are not accessible.
Name of the bacterial strains that is of lactobacillus species:-
Bacillus mycoids
Bacillus thiringiensis
Bacillus subtitis
Bacillus megatorium
Lactobacillus plantarum
Lactobacillus brevis
Lactobacillus casei
Lactobacillus bifidus
Lactobacillus fermentum
Lactobacillus bulgaricus
Bacillus sp.
The bacteria in the sachet were:-
Lactobacillus acidophilus
Lactobacillus rhamnosus
Biffidobacterum longum
Biffido bacterium bifidum
Sachharomyces bouladill
Frucio oligo sachharides
These bacteria have been reported to produce high amount of bioplastics (PHB) and are also
probiotic .therefore, these are safe organism.
Lactobacillus acidophilus: -L.actobacillus acidophilus is a types of gram positive microscopic
organisms in the class Lactobacillus. L. acidophilus is a homofermentative, microaerophilic animal
types, aging sugars into lactic corrosive, and develops promptly at rather low philus happens
characteristically in the human and creature gastrointestinal tract and mouth. A few strains of L.
acidophilus may be considered to have probiotic characteristics. These strains are financially
utilized as a part of numerous dairy items, infrequently together with Streptococcus thermophilus
and Lactobacillus delbrueckii subsp. bulgaricus in the generation of acidophilus-sort yogurt.
Lactobacillus acidophilus
Methods to find out the yield of PHB:-
Gas chromatography
High performance liquid chromatography
Gas chromatography:-
This is a technique is very useful for separating and analyzing compounds which vaporize because
of decomposition. It has its application in the field of testing purity of a substance as well as
separating various components in a mixture. It helps identify required compound too which is the
property that can be used to find out the yield of PHB. It can also assist in making a pure compound
from a mixture of substances.
Its mechanism is that it has a carrier gas as its mobile phase which can be helium or nitrogen (inert).
Stationary phase consists of a layer of liquid or polymer kept on a solid support which is inert. This
setup can be kept within a column. Retention time gives information about each compound to be
separated.
HIGH PERFORMANCE LIQUID CHROMATOGRAPH:-
It has same uses as gas chromatography but it has an additional advantage of quantifying each
component too. It consists of a pump and a pressurized liquid sample is passed through a column
consisting of solid adsorbent. That contains the sample to be separated. The components of the
mixture have different reaction rates with the solid adsorbent. Thus different components with ease.
It has high pressure at which it operates because of which it is different from gas chromatography.
MATERIALS AND METHODS:-
Isolation of microbial structure:-
Isolation of E.coli was done from garden soil using serial dilution method. Each bacterial colony was
isolated and maintained in slant culture tubes containing LB agar medium. Liquid culture was prepared
by inoculating a loop full of bacterial cells in 100 ml nutrient broth medium and incubating the culture
overnight at 37 °C in an incubator shaker set at 200 rpm. The inoculum culture age was 14-16 h..
Batch cultivation of E.coli cells in a Stirred tank bioreactor:-
The composition of growth culture medium of E.coli has been shown in Table 2. The 5 L fermentor
(BIOSTAT B+ Twin Bioreactor, Sartorius-Germany) was inoculated with 110 ml culture from 14 -16 h
old primary culture of E.coli strain PC-I. Total medium volume in the 5 l fermentor was 2.1 L. The
bioreactor was maintained at a constant incubation temperature of 37 °C. The batch cultivation was
continued for incubation time of 12h. The Stirred tank reactor impeller was maintained at constant
rotational speed of 220 rpm. The pH of the culture was maintained at 6.8 using bio controller.
S.No. Component Concentration (g/l)1. Yeast extract 1.02. Citric acid 1.73. KH2PO4 6.84. Na2HPO4 8.95. Glucose 3.06. MgSO4 0.2
Measurement of cell optical density:-
The optical density measurement on spectrophotometer is based on light scattering. In
spectrophotometerwith Beer Lambert’s law, the broth sample should be suitably diluted so that its
OD600nm is between 0.1-0.4. The OD of undiluted sample can be obtained by multiplying diluted sample
can be obtained by multiplying diluted sample OD with the dilution factor.
Measurement of cell concentration:-
For the measurement of Cell concentration 50ml empty falcon tube was weighed. 1ml broth sample was
added to it and centrifuged for 5 minutes at 10,000 rpm. The supernatant was decanted and the residue
was dried in tube at 400C under vacuum for 24h. Thereafter the tube was placed in a desiccators for
cooling. After 30 minutes, the tube was weighed again. Then weight of empty tube was subtracted from
it. This gives us cell dry weight in (g/l) of the sample broth.
Measurement of glucose concentration:-
Measurement of glucose concentration was done using di-nitrosalicylic acid (DNS) reagent. Preparation
of standard curve for glucose was done in the range of sugar estimation by the DNS method that is 0.1-
4.0 mg/ml sugar in sample. 3ml DNS reagent was added to all the tubes, and the tubes were kept in
boiling water bath for exactly 5 minutes. Thereafter, all the tubes were cooled under running tap water.
20 ml distilled water was added to all the tubes and mixed thoroughly. Absorbance at 540 nm
wavelength was found on a spectrophotometer using sixth tube as a blank. A plot of OD vs Sugar
concentration (mg/ml) was prepared.
Measurement of glucose:
For measurement of glucose in the sample, we centrifuged the sample and diluted the supernatant. DNS
treatment was performed and the OD was read against a blank. The sugar standard curve was used for
calculation of glucose concentration in the sample of fermentation broth.
Estimation of polyhydroxybutyrate:
1ml freshly prepared phosphate buffer saline was added to the stored cell pellet of E.coli. After
mixing well the cell pellet was centrifuged for 10 minutes at 15000 rpm at room temperature. PBS
was slowly removed from the falcon tube to achieve the target of washing the cell pellet. Then the
cell pellet was air dried for 20 minutes. After that 1ml concentrated H2SO4 was added and boiled
at 100 degree Celsius for 20 minutes. After cooling the sample to room temperature dilution was
prepared from 10microlitre/ml to 100microlitre/ml. Absorbance at 235nm was taken for all the
dilutions using distilled water as blank.
Standard curve for polyhydroxybutyrate:-
200mg PHB was dissolved in 1 ml of concentrated H2SO4 and boiled for 10 minutes. In this event,
PHB is converted into crotonic acid and cool it. Concentration of the solution is 200mg/ml
(crotonic acid).solution was used as stock solution. Now prepared a dilution from 10mg/ml
distilled water used as a diluent.took absorbance at 235nm, distilled water is used as a blank.
Stocks solution is stored at 4c for further use.
Result and description:
Isolation of microbial structure:-
Fig1
Cultivation of E.coli cells in a Stirred tank
Bioreactor:-
Stirred tank reactor is a preferred mode of cultivation which provides adequate oxygen supply to
growing aerobic bacterial cultures. E. coli cells were cultivated in a 5-L stirred tank reactor for 17 hours
till stationary phase. Biomass concentration and substrate concentration were estimated at an interval of
30 minutes. The specific growth rate was found out to be 0.065 which is the slope of graph of ln(DW) vs
time.
The average growth yield coefficient was found out to be 0.0725 which can be calculated from the
formula:
YX/S = (XF-X0)/ (SF-S0)
Where XF& SF represent cell dry weight and substrate concentration at the end of fermentation
respectively, and X0 and S0 represent dry cell weight and substrate concentration at the beginning of the
fermentation process.
Fig2:- OD vs TIME
Standard Curve for polyhydroxybutyrate:
The absorbance readings table at 235nm for different concentrations of Standard sample of PHB is as
follows:-
Concentration in
ug/ml Absorbance at 235nm Ave.blank 0 0 0 0
10 0.028 0.028 0.029
0.02833
320 0.058 0.058 0.058 0.05830 0.098 0.098 0.098 0.098
40 0.135 0.136 0.135
0.13533
3
50 0.171 0.172 0.172
0.17166
760 0.222 0.222 0.222 0.22270 0.265 0.265 0.265 0.265
80 0.318 0.319 0.319
0.31866
7
90 0.349 0.349 0.348
0.34866
7
100 0.401 0.403 0.403
0.40233
3
110 0.423 0.423 0.424
0.42333
3
120 0.437 0.437 0.438
0.43733
3
130 0.451 0.451 0.452
0.45133
3140 0.478 0.476 0.477 0.477150 0.535 0.536 0.535 0.53533
3
160 0.564 0.564 0.565
0.56433
3
170 0.59 0.589 0.59
0.58966
7180 0.632 0.632 0.632 0.632
190 0.664 0.664 0.663
0.66366
7200 0.696 0.695 0.697 0.696
Estimation of polyhydroxybutyrate:
Absorbance readings for different dilutions of E.coli at 235nm:-
DILUTION(microliter/litre) ABSORBANCE20 0.095, 0.094, 0.095
40 0.205, 0.205,0.20560 0.315, 0.314,0.31480 0.360,0.360,0361100 0.428,0.428,0.428
Conclusion
In this thesis, PHB was produced by serial dilution method. We
estimated PHB and plotted standard curve of PHB. The dry cell
weight was taken 0.247gm and estimation of PHB was 11 mg.The
yield of PHB was 5.725mg/DW.
reference
▸ Braunegg et al., 1978G. Braungg, B. Sonnleitner, R.M. LaffertyArapid gas chromatographic
method for the determination of poly- β-hydroxybutyric acid in microbial biomass
▸ Eur. J. Appl. Microbiol. Biotechnol., 6 (1978), pp. 29–37
▸ Impact of Ralstonia eutropha's Poly(3-Hydroxybutyrate) (PHB) Depolymerases and Phasins on
PHB Storage in Recombinant Escherichia coli Appl. Environ. Microbiol. 2014 80: 7702-7709.
▸ Microbial production of polyhydroxybutyrate, a biodegradable plastic using agro-industrial
waste products,Alyaa Hamieh*, Zakia Olama and Hanafi Holail , Global Advanced Research
Journal of Microbiology (ISSN: 2315-5116), 2013